Human HNEAA81 receptor

ABSTRACT

HNEAA81 polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are screening methods for identifying agonists and antagonists of the interaction of the HNEAA81 receptor and its ligands in the design of protocols for the treatment of infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HIV-1 or HIV-2; pain; cancers; anorexia; bulimia; asthma; Parkinson&#39;s disease; acute heart failure; hypotension; hypertension; urinary retention; osteoporosis; angina pectoris; myocardial infarction; ulcers; asthma; allergies; benign prostatic hypertrophy; and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington&#39;s disease or Gilles dela Tourett&#39;s syndrome, among others and diagnostic assays for such conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U.S.application Ser. No. 08/956,975, filed on Oct. 23, 1997, the contents ofwhich are herein incorporated by reference in their entirety.

FIELD OF INVENTION

This invention relates to newly identified polynucleotides, polypeptidesencoded by them and to the use of such polynucleotides and polypeptides,and to their production. More particularly, the polynucleotides andpolypeptides of the present invention relate to the G-protein coupledreceptor family, hereinafter referred to as HNEAA81. The invention alsorelates to inhibiting or activating the action of such polynucleotidesand polypeptides.

BACKGROUND OF THE INVENTION

It is well established that many medically significant biologicalprocesses are mediated by proteins participating in signal transductionpathways that involve G-proteins and/or second messengers, e.g., cAMP(Lefkowitz, Nature, 1991, 351:353-354). Herein these proteins arereferred to as proteins participating in pathways with G-proteins or PPGproteins. Some examples of these proteins include the GPC receptors,such as those for adrenergic agents and dopamine (Kobilka, et al., Proc.Natl Acad. Sci., USA, 1987, 84:46-50; Kobilka, et al., Science, 1987,238:650-656; Bunzow, et al., Nature, 1988, 336:783-787), G-proteinsthemselves, effector proteins, e.g., phospholipase C, adenyl cyclase,and phosphodiesterase, and actuator proteins, e.g. protein kinase A andprotein kinase C (Simon, et al., Science, 1991, 252:802-8).

For example, in one form of signal transduction, the effect of hormonebinding is activation of the enzyme, adenylate cyclase, inside the cell.Enzyme activation by hormones is dependent on the presence of thenucleotide, GTP. GTP also influences hormone binding. A G-proteinconnects the hormone receptor to adenylate cyclase. G-protein was shownto exchange GTP for bound GDP when activated by a hormone receptor. TheGTP-carrying form then binds to activated adenylate cyclase. Hydrolysisof GTP to GDP, catalyzed by the G-protein itself, returns the G-proteinto its basal, inactive form. Thus, the G-protein serves a dual role, asan intermediate that relays the signal from receptor to effector, and asa clock that controls the duration of the signal.

The membrane protein gene superfamily of G-protein coupled receptors hasbeen characterized as having seven putative transmembrane domains. Thedomains are believed to represent transmembrane α-helices connected byextracellular or cytoplasmic loops. G-protein coupled receptors includea wide range of biologically active receptors, such as hormone, viral,growth factor and neuroreceptors.

G-protein coupled receptors (otherwise known as 7TM receptors) have beencharacterized as including these seven conserved hydrophobic stretchesof about 20 to 30 amino acids, connecting at least eight divergenthydrophilic loops. The G-protein family of coupled receptors includesdopamine receptors which bind to neuroleptic drugs used for treatingpsychotic and neurological disorders. Other examples of members of thisfamily include, but are not limited to, calcitonin, adrenergic,endothelin, cAMP, adenosine, muscarinic, acetylcholine, serotonin,histamine, thrombin, kinin, follicle stimulating hormone, opsins,endothelial differentiation gene-1, rhodopsins, odorant, andcytomegalovirus receptors.

Most G-protein coupled receptors have single conserved cysteine residuesin each of the first two extracellular loops which form disulfide bondsthat are believed to stabilize functional protein structure. The 7transmembrane regions are designated as TM1, TM2, TM3, TM4, TM5, TM6,and TM7. TM3 has been implicated in signal transduction.

Phosphorylation and lipidation (palmitylation or farnesylation) ofcysteine residues can influence signal transduction of some G-proteincoupled receptors. Most G-protein coupled receptors contain potentialphosphorylation sites within the third cytoplasmic loop and/or thecarboxy terminus. For several G-protein coupled receptors, such as theβ-adrenoreceptor, phosphorylation by protein kinase A and/or specificreceptor kinases mediates receptor desensitization.

For some receptors, the ligand binding sites of G-protein coupledreceptors are believed to comprise hydrophilic sockets formed by severalG-protein coupled receptor transmembrane domains, said socket beingsurrounded by hydrophobic residues of the G-protein coupled receptors.The hydrophilic side of each G-protein coupled receptor transmembranehelix is postulated to face inward and form a polar ligand binding site.TM3 has been implicated in several G-protein coupled receptors as havinga ligand binding site, such as the TM3 aspartate residue. TM5 serines, aTM6 asparagine and TM6 or TM7 phenylalanines or tyrosines are alsoimplicated in ligand binding.

G-protein coupled receptors can be intracellularly coupled byheterotrimeric G-proteins to various intracellular enzymes, ion channelsand transporters (see, Johnson, et al., Endoc. Rev., 1989, 10:317-331)Different G-protein α-subunits preferentially stimulate particulareffectors to modulate various biological functions in a cell.Phosphorylation of cytoplasmic residues of G-protein coupled receptorshas been identified as an important mechanism for the regulation ofG-protein coupling of some G-protein coupled receptors. G-proteincoupled receptors are found in numerous sites within a mammalian host.

Over the past 15 years, nearly 350 therapeutic agents targeting 7transmembrane (7 TM) receptors have been successfully introduced intothe market.

This indicates that these receptors have an established, proven historyas therapeutic targets. Clearly there is a need for identification andcharacterization of further receptors which can play a role inpreventing, ameliorating or correcting dysfunctions or diseases,including, but not limited to, infections such as bacterial, fungal,protozoan and viral infections, particularly infections caused by HIV-1or HIV-2; pain; cancers; anorexia; bulimia; asthma; Parkinson's disease;acute heart failure; hypotension; hypertension; urinary retention;osteoporosis; angina pectoris; myocardial infarction; ulcers; asthma;allergies; benign prostatic hypertrophy; and psychotic and neurologicaldisorders, including anxiety, schizophrenia, manic depression, delirium,dementia, severe mental retardation and dyskinesias, such asHuntington's disease or Gilles dela Tourett's syndrome.

SUMMARY OF THE INVENTION

In one aspect, the invention relates to HNEAA81 polypeptides andrecombinant materials and methods for their production. Another aspectof the invention relates to methods for using such HNEAA81 polypeptidesand polynucleotides. Such uses include the treatment of infections suchas bacterial, fungal, protozoan and viral infections, particularlyinfections caused by HIV-1 or HIV-2; pain; cancers; anorexia; bulimia;asthma; Parkinson's disease; acute heart failure;hypotension;hypertension; urinary retention; osteoporosis; angina pectoris;myocardial infarction; ulcers; asthma; allergies; benign prostatichypertrophy; and psychotic and neurological disorders, includinganxiety, schizophrenia, manic depression, delirium, dementia, severemental retardation and dyskinesias, such as Huntington's disease orGilles dela Tourett's syndrome, among others. In still another aspect,the invention relates to methods to identify agonists and antagonistsusing the materials provided by the invention, and treating conditionsassociated with HNEAA81 imbalance with the identified compounds.

In still another aspect, the invention relates to methods to identifyagonists and antagonists using the materials provided by the invention,and treating conditions associated with HNEAA81 imbalance with theidentified compounds. In particular, the preferred method foridentifying agonist or antagonist of HNEAA81 receptor of the presentinvention comprises:

contacting a cell expressing on the surface thereof the receptor, saidreceptor being associated with a second component capable of providing adetectable signal in response to the binding of a compound to saidreceptor, with a compound to be screened under conditions to permitbinding to the receptor; and

determining whether the compound binds to and activates or inhibits thereceptor by measuring the level of a signal generated from theinteraction of the compound with the receptor.

In a further preferred embodiment, the method further comprisesconducting the identification of agonist or antagonist in the presenceof labeled or unlabeled di-adenosine hexaphosphate (hereinafter referredto as "AP6A"), di-adenosine pentaphosphate (hereinafter referred to as"APSA"), or deoxy-uridine di-phosphate (hereinafter referred to as"d-LUDP").

In another embodiment of the method for identifying agonist orantagonist of a HNEAA81 receptor of the present invention comprises:

determining the inhibition of binding of a ligand to cells which havethe receptor on the surface thereof, or to cell membranes containing thereceptor, in the presence of a candidate compound under conditions topermit binding to the receptor, and determining the amount of ligandbound to the receptor, such that a compound capable of causing reductionof binding of a ligand is an agonist or antagonist. Preferably, theligand is AP6A, AP5A, or d-UDP. Yet more preferably, AP6A, AP5A, ord-UDP is labeled.

Yet another aspect of the invention relates to diagnostic assays fordetecting diseases associated with inappropriate HNEAA81 activity orlevels.

DESCRIPTION OF THE INVENTION

Definitions

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein.

"HNEAA81" refers, among others, to a polypeptide comprising the aminoacid sequence set forth in SEQ ID NO:2, or an allelic variant thereof.

"Receptor Activity" or "Biological Activity of the Receptor" refers tothe metabolic or physiologic function of said HNEAA81 including similaractivities or improved activities or these activities with decreasedundesirable side-effects. Also included are antigenic and immunogenicactivities of said HNEAA81.

"HNEAA81 gene" refers to a polynucleotide comprising the nucleotidesequence set forth in SEQ ID NO:1 or allelic variants thereof and/ortheir complements.

"AP6A" refers to di-adenosine hexaphosphate, which has the followingstructure: ##STR1##

"AP5A" refers to di-adenosine pentaphosphate, which has the followingstructure: ##STR2##

"d-UDP" refers to deoxy-uridine di-phosphate, which has the followingstructure: ##STR3##

"Antibodies" as used herein includes polyclonal and monoclonalantibodies, chimeric, single chain, and humanized antibodies, as well asFab fragments, including the products of an Fab or other immunoglobulinexpression library.

"Isolated" means altered "by the hand of man" from the natural state. Ifan "isolated" composition or substance occurs in nature, it has beenchanged or removed from its original environment, or both. For example,a polynucleotide or a polypeptide naturally present in a living animalis not "isolated," but the same polynucleotide or polypeptide separatedfrom the coexisting materials of its natural state is "isolated", as theterm is employed herein.

"Polynucleotide" generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. "Polynucleotides" include, without limitation single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, "polynucleotide" refers to triple-stranded regions comprisingRNA or DNA or both RNA and DNA. The term polynucleotide also includesDNAs or RNAs containing one or more modified bases and DNAs or RNAs withbackbones modified for stability or for other reasons. "Modified" basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications has been made to DNA and RNA; thus,"polynucleotide" embraces chemically, enzymatically or metabolicallymodified forms of polynucleotides as typically found in nature, as wellas the chemical forms of DNA and RNA characteristic of viruses andcells. "Polynucleotide" also embraces relatively short polynucleotides,often referred to as oligonucleotides.

"Polypeptide" refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds, i.e., peptide isosteres. "Polypeptide" refers to both shortchains, commonly referred to as peptides, oligopeptides or oligomers,and to longer chains, generally referred to as proteins. Polypeptidesmay contain amino acids other than the 20 gene-encoded amino acids."Polypeptides" include amino acid sequences modified either by naturalprocesses, such as posttranslational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched as a result of ubiquitination, and they maybe cyclic, with or without branching. Cyclic, branched and branchedcyclic polypeptides may result from posttranslation natural processes ormay be made by synthetic methods. Modifications include acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cystine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination. See, for instance, PROTEINS--STRUCTURE AND MOLECULARPROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, NewYork, 1993 and Wold, F., Posttranslational Protein Modifications:Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENTMODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York,1983; Seifter, et al., "Analysis for protein modifications andnonprotein cofactors", Meth Enzymol (1990) 182:626-646 and Rattan etal., "Protein Synthesis: Posttranslational Modifications and Aging", AnnNY Acad Sci (1992) 663:48-62.

"Variant" as the term is used herein, is a polynucleotide or polypeptidethat differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. A variant of a polynucleotide or polypeptide may be a naturallyoccurring such as an allelic variant, or it may be a variant that is notknown to occur naturally. Non-naturally occurring variants ofpolynucleotides and polypeptides may be made by mutagenesis techniquesor by direct synthesis.

"Identity," as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, "identity" also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. "Identity" and "similarity" can be readilycalculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing. Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073(1988). Preferred methods to determine identity are designed to give thelargest match between the sequences tested. Methods to determineidentity and similarity are codified in publicly available computerprograms. Preferred computer program methods to determine identity andsimilarity between two sequences include, but are not limited to, theGCG program package (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec.Biol. 215: 403-410 (1990). The BLAST X program is publicly availablefrom NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). The well known Smith Waterman algorithm may also be usedto determine identity.

Preferred parameters for polypeptide sequence comparison include thefollowing:

1) Algorithm: Needleman and Wunsch, J Mol. Biol. 48: 443-453 (1970)Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl.Acad. Sci. USA. 89:10915-10919 (1992)

Gap Penalty: 12

Gap Length Penalty: 4

A program useful with these parameters is publicly available as the"gap" program from Genetics Computer Group, Madison Wis. Theaforementioned parameters are the default parameters for polypeptidecomparisons (along with no penalty for end gaps).

Preferred parameters for polynucleotide comparison include thefollowing:

1) Algorithm: Needleman and Wunsch, J. Mol BioL. 48: 443-453 (1970)

Comparison matrix: matches=+10, mismatch=0

Gap Penalty: 50

Gap Length Penalty: 3

A program useful with these parameters is publicly available as the"gap" program from Genetics Computer Group, Madison Wis. Theaforementioned parameters are the default parameters for polynucleotidecomparisons.

Preferred polynucleotide embodiments further include an isolatedpolynucleotide comprising a polynucleotide having at least a 50, 60, 70,80, 85, 90, 95, 97 or 100% identity to a polynucleotide referencesequence of SEQ ID NO:1, wherein said reference sequence may beidentical to the sequence of SEQ ID NO:1 or may include up to a certaininteger number of nucleotide alterations as compared to the referencesequence, wherein said alterations are selected from the groupconsisting of at least one nucleotide deletion, substitution, includingtransition and transversion, or insertion, and wherein said alterationsmay occur at the 5' or 3' terminal positions of the reference nucleotidesequence or anywhere between those terminal positions, interspersedeither individually among the nucleotides in the reference sequence orin one or more contiguous groups within the reference sequence, andwherein said number of nucleotide alterations is determined bymultiplying the total number of nucleotides in SEQ ID NO:1 by thenumerical percent of the respective percent identity and subtractingthat product from said total number of nucleotides in SEQ ID NO:1, or:

    n.sub.n ≦x.sub.n -(x.sub.n.y),

wherein n_(n) is the number of nucleotide alterations, x_(n) is thetotal number of nucleotides in SEQ ID NO:1, and y is 0.50 for 50%, 0.60for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95for 95%, 0.97 for 97% or 1.00 for 100%, and wherein any non-integerproduct of x_(n) and y is rounded down to the nearest integer prior tosubtracting it from x_(n). Alterations of a polynucleotide sequenceencoding the polypeptide of SEQ ID NO:2 may create nonsense, missense orframeshift mutations in this coding sequence and thereby alter thepolypeptide encoded by the polynucleotide following such alterations.

Preferred polypeptide embodiments further include an isolatedpolypeptide comprising a polypeptide having at least a 50, 60, 70, 80,85, 90, 95, 97 or 100% identity to a polypeptide reference sequence ofSEQ ID NO:2, wherein said reference sequence may be identical to thesequence of SEQ ID NO:2 or may include up to a certain integer number ofamino acid alterations as compared to the reference sequence, whereinsaid alterations are selected from the group consisting of at least oneamino acid deletion, substitution, including conservative andnon-conservative substitution, or insertion, and wherein saidalterations may occur at the amino- or carboxy-terminal positions of thereference polypeptide sequence or anywhere between those terminalpositions, interspersed either individually among the amino acids in thereference sequence or in one or more contiguous groups within thereference sequence, and wherein said number of amino acid alterations isdetermined by multiplying the total number of amino acids in SEQ ID NO:2by the numerical percent of the respective percent identity andsubtracting that product from said total number of amino acids in SEQ IDNO:2, or:

    n.sub.a ≦x.sub.a -(x.sub.a.y),

wherein n_(a) is the number of amino acid alterations, x_(a) is thetotal number of amino acids in SEQ ID NO:2, and y is 0.50 for 50%, 0.60for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95for 95%, 0.97 for 97% or 1.00 for 100%, and wherein any non-integerproduct of x_(a) and y is rounded down to the nearest integer prior tosubtracting it from x_(a).

Polypeptides of the Invention

In one aspect, the present invention relates to HNEAA81 polypeptides (orHNEAA81 proteins). The HNEAA81 polypeptides include the polypeptide ofSEQ ID NO:2; as well as polypeptides comprising the amino acid sequenceof SEQ ID NO:2; and polypeptides comprising the amino acid sequencewhich have at least 80% identity to that of SEQ ID NO:2 over its entirelength, and still more preferably at least 90% identity, and even stillmore preferably at least 95% identity to SEQ ID NO:2. Furthermore, thosewith at least 97-99% are highly preferred. Also included within HNEAA81polypeptides are polypeptides having the amino acid sequence which haveat least 80% identity to the polypeptide having the amino acid sequenceof SEQ ID NO:2 over its entire length, and still more preferably atleast 90% identity, and even still more preferably at least 95% identityto SEQ ID NO:2. Furthermore, those with at least 97-99% are highlypreferred. Preferably, HNEAA81 polypeptides exhibit at least onebiological activity of the receptor.

The HNEAA81 polypeptides may be in the form of the "mature" protein ormay be a part of a larger protein such as a fusion protein. It is oftenadvantageous to include an additional amino acid sequence which containssecretory or leader sequences, pro-sequences, sequences which aid inpurification such as multiple histidine residues, or an additionalsequence for stability during recombinant production.

Fragments of the HNEAA81 polypeptides are also included in theinvention. A fragment is a polypeptide having an amino acid sequencethat entirely is the same as part, but not all, of the amino acidsequence of the aforementioned HNEAA81 polypeptides. As with HNEAA81polypeptides, fragments may be "free-standing," or comprised within alarger polypeptide of which they form a part or region, most preferablyas a single continuous region. Representative examples of polypeptidefragments of the invention, include, for example, fragments from aboutamino acid number 1-20, 21-40, 41-60, 61-80, 81-100, and 101 to the endof the HNEAA81 polypeptide. In this context "about" includes theparticularly recited ranges larger or smaller by several, 5, 4, 3, 2 or1 amino acid at either extreme or at both extremes.

Preferred fragments include, for example, truncation polypeptides havingthe amino acid sequence of HNEAA81 polypeptides, except for deletion ofa continuous series of residues that includes the amino terminus, or acontinuous series of residues that includes the carboxyl terminus ordeletion of two continuous series of residues, one including the aminoterminus and one including the carboxyl terminus. Also preferred arefragments characterized by structural or functional attributes such asfragments that comprise alpha-helix and alpha-helix forming regions,beta-sheet and beta-sheet-forming regions, turn and turn-formingregions, coil and coil-forming regions, hydrophilic regions, hydrophobicregions, alpha amphipathic regions, beta amphipathic regions, flexibleregions, surface-forming regions, substrate binding region, and highantigenic index regions. Other preferred fragments are biologicallyactive fragments. Biologically active fragments are those that mediatereceptor activity, including those with a similar activity or animproved activity, or with a decreased undesirable activity. Alsoincluded are those that are antigenic or immunogenic in an animal,especially in a human.

Preferably, all of these polypeptide fragments retain the biologicalactivity of the receptor, including antigenic activity. Variants of thedefined sequence and fragments also form part of the present invention.Preferred variants are those that vary from the referents byconservative amino acid substitutions--i.e., those that substitute aresidue with another of like characteristics. Typical such substitutionsare among Ala, Val, Leu and Ile; among Ser and Thr; among the acidicresidues Asp and Glu; among Asn and Gln; and among the basic residuesLys and Arg; or aromatic residues Phe and Tyr. Particularly preferredare variants in which several, 5 10, 1-S, or 1-2 amino acids aresubstituted, deleted, or added in any combination.

The HNEAA81 polypeptides of the invention can be prepared in anysuitable manner. Such polypeptides include isolated naturally occurringpolypeptides, recombinantly produced polypeptides, syntheticallyproduced polypeptides, or polypeptides produced by a combination ofthese methods. Means for preparing such polypeptides are well understoodin the art.

Polynucleotides of the Invention

Another aspect of the invention relates to HNEAA81 polynucleotides.HNEAA81 polynucleotides include isolated polynucleotides which encodethe HNEAA81 polypeptides and fragments, and polynucleotides closelyrelated thereto. More specifically, the HNEAA81 polynucleotides of theinvention include a polynucleotide comprising the nucleotide sequencecontained in SEQ ID NO:1 encoding an HNEAA81 polypeptide of SEQ ID NO:2,and polynucleotide having the particular sequence of SEQ ID NO:1.HNEAA81 polynucleotides further include a polynucleotide comprising anucleotide sequence that has at least 80% identity over its entirelength to a nucleotide sequence encoding the HNEAA81 polypeptide of SEQID NO:2, and a polynucleotide comprising a nucleotide sequence that isat least 80% identical to that of SEQ ID NO:1 over its entire length. Inthis regard, polynucleotides at least 90% identical are particularlypreferred, and those with at least 95% are especially preferred.Furthermore, those with at least 97% are highly preferred and those withat least 98-99% are most highly preferred, with at least 99% being themost preferred. Also included under HNEAA81 polynucleotides are anucleotide sequence which has sufficient identity to a nucleotidesequence contained in SEQ ID NO:1 to hybridize under conditions useablefor amplification or for use as a probe or marker. The invention alsoprovides polynucleotides which are complementary to such HNEAA81polynucleotides.

HNEAA81 of the invention is structurally related to other proteins oftheG-protein coupled receptor family, as shown by the results ofsequencing the cDNA of Table 1 (SEQ ID NO:1) encoding human HNEAA8 1.The cDNA sequence of SEQ ID NO:1 contains an open reading frame(nucleotide number 98 to 1096) encoding a polypeptide of 333 amino acids(SEQ ID NO:2). The amino acid sequence of Table 2 (SEQ ID NO:2) hasabout 74.914% identity in 293 amino acid residues with human G-proteincoupled receptor; GPR3 (Geneseqp patent database, Accession # W04246,Bult, C. J., et al., Dec. 13, 1996). Furthermore, HNEAA81 (SEQ ID NO:2)is 28.0% identical (FASTA, Swisspro databse) to platelet activatingfactor receptor over 293 amino acid residues (Honda, et al., Nature349:342-346, 1991). Furthermore, HNEAA81 (SEQ ID No: 2) is 25.6%identical to thrombin receptor over 305 amino acid residues (Accession#P47749,Turck, et al, Nature 368: 648-651, 1994). Furthermore, HNEAA81(SEQ ID NO:2) is 26.5% identical to EBV-Induced G-protein coupledreceptor, EBI2 over 313 amino acid residues (Accession # P32249,Elliott, et al., J. Virol. 67: 2209-2220, 1993). The nucleotide sequenceof Table 1 (SEQ ID NO:1) has about 96% identity in 1124 nucleotideresidues with human G-protein coupled receptor (Geneseqn patentdatabase, Accession #T33904, Bult,C. J. et al, Dec. 13, 1996).Furthermore, HNEAA81 (SEQ ID No:1) is 56.47% identical (BLAST usingGenebank database) to human mRNA for KIA0001 gene over 850 nucleotideresidues (Accession #D13626, Nomura, et al., Unpublished, 1994). Thus,HNEAA81 polypeptides and polynucleotides of the present invention areexpected to have, inter alia, similar biological functions/properties totheir homologous polypeptides and polynucleotides, and their utility isobvious to anyone skilled in the art.

                                      TABLE 1.sup.a                               __________________________________________________________________________       1 TCTGGTTTTT AAAAAATAGC ATTTGAAAAT CATGAAGGGC TTTTTGTTTT                     51 CTTTTGTTTG TATATATGTT TATTGGTAAC AGGTGACACT GGAAGCAATG                    101 AACACCACAG TGATGCAAGG CTTCAACAGA TCTGAGCGGT GCCCCAGAGA                    151 CACTCGGATA GTACAGCTGG TATTCCCAGC CCTCTACACA GTGGTTTTCT                    201 TGACCGGCAT CCTGCTGAAT ACTTTGGCTC TGTGGGTGTT TGTTCACATC                    251 CCCAGCTCCT CCACCTTCAT CATCTACCTC AAAAACACTT TGGTGGCCGA                    301 CTTGATAATG ACACTCATGC TTCCTTTCAA AATCCTCTCT GACTCACACC                    351 TGGCACCCTG GCAGCTCAGA GCTTTTGTGT GTCGTTTTTC TTCGGTGATA                    401 TTTTATGAGA CCATGTATGT GGGCATCGTG CTGTTAGGGC TCATAGCCTT                    451 TGACAGATTC CTCAAGATCA TCAGACCTTT GAGAAATATT TTTCTAAAAA                    501 AACCTGTTTT TGCAAAAACG GTCTCAATCT TCATCTGGTT CTTTTTGTTC                    551 TTCATCTCCC TGCCAAATAC GATCTTGAGC AACAAGGAAG CAACACCATC                    601 GTCTGTGAAA AAGTGTGCTT CCTTAAAGGG GCCTCTGGGG CTGAAATGGC                    651 ATCAAATGGT AAATAACATA TGCCAGTTTA TTTTCTGGAC TGTTTTTATC                    701 CTAATGCTTG TGTTTTATGT GGTTATTGCA AAAAAAGTAT ATGATTCTTA                    751 TAGAAAGTCC AAAAGTAAGG ACAGAAAAAA CAACAAAAAG CTGGAAGGCA                    801 AAGTATTTGT TGTCGTGGCT GTCTTCTTTG TGTGTTTTGC TCCATTTCAT                    851 TTTGCCAGAG TTCCATATAC TCACAGTCAA ACCAACAATA AGACTGACTG                    901 TAGACTGCAA AATCAACTGT TTATTGCTAA AGAAACAACT CTCTTTTTGG                    951 CAGCAACTAA CATTTGTATG GATCCCTTAA TATACATATT CTTATGTAAA                   1001 AAATTCACAG AAAAGCTACC ATGTATGCAA GGGAGAAAGA CCACAGCATC                   1051 AAGCCAAGAA AATCATAGCA GTCAGACAGA CAACATAACC TTAGGCTGAC                   1101 AACTGTACAT AGGGTTAACT TCTA                                               __________________________________________________________________________     .sup.a A nucleotide sequence of a human HNEAA81 (SEQ ID NO: 1).   }{0160 

                                      TABLE 2.sup.b                               __________________________________________________________________________      1 MNTTVMQGFN RSERCPRDTR IVQLVFPALY TVVFLTGILL NTLALWVFVH                     51 IPSSSTFIIY LKNTLVADLI MTLMLPFKIL SDSHLAPWQL RAFVCRFSSV                    101 IFYETMYVGI VLLGLIAFDR FLKIIRPLRN IFLKKPVFAK TVSIFIWFFL                    151 FFISLPNTIL SNKEATPSSV KKCASLKGPL GLKWHQMVNN ICQFIFWTVF                    201 ILMLVFYVVI AKKVYDSYRK SKSKDRKNNK KLEGKVFVVV AVFFVCFAPF                    251 HFARVPYTHS QTNNKTDCRL QNQLFIAKET TLFLAATNIC MDPLIYIFLC                    301 KKFTEKLPCM QGRKTTASSQ ENHSSQTDNI TLG                                      __________________________________________________________________________     .sup.b An amino acid sequence of a human HNEAA81 (SEQ ID NO:2).          

One polynucleotide of the present invention encoding HNEAA81 may beobtained using standard cloning and screening, from a cDNA libraryderived from mRNA in cells ofihuman brain, leukocyte, and lung using theexpressed sequence tag (EST) analysis (Adams, M. D., et al. Science(1991) 252:1651-1656; Adams, M. D., et al., Nature, (1992) 355:632-634;Adams, M. D., et al., Nature (1995) 377 Supp:3-174). Polynucleotides ofthe invention can also be obtained from natural sources such as genomicDNA libraries or can be synthesized using well known and commerciallyavailable techniques.

The nucleotide sequence encoding the HNEAA81 polypeptide of SEQ ID NO:2may be identical to the polypeptide encoding sequence contained in Table1 (nucleotide number 98 to 1096 of SEQ ID NO:1), or it may be asequence, which as a result of the redundancy (degeneracy) of thegenetic code, also encodes the polypeptide of SEQ ID NO:2.

When the polynucleotides of the invention are used for the recombinantproduction of an HNEAA81 polypeptide, the polynucleotide may include thecoding sequence for the mature polypeptide or a fragment thereof, byitself; the coding sequence for the mature polypeptide or fragment inreading frame with other coding sequences, such as those encoding aleader or secretory sequence, a pre-, or pro- or prepro- proteinsequence, or other fusion peptide portions. For example, a markersequence which facilitates purification of the fused polypeptide can beencoded. In certain preferred embodiments of this aspect of theinvention, the marker sequence is a hexa-histidine peptide, as providedin the pQE vector (Qiagen, Inc.) and described in Gentz, et al., ProcNatl Acad Sci USA (1989) 86:821-824, or is an HA tag. The polynucleotidemay also contain non-coding 5' and 3' sequences, such as transcribed,non-translated sequences, splicing and polyadenylation signals, ribosomebinding sites and sequences that stabilize mRNA.

Further preferred embodiments are polynucleotides encoding HNEAA81variants comprising the amino acid sequence of the HNEAA81 polypeptideof Table 2 (SEQ ID NO:2) in which several, 5-10, 1-5, 1-3, 1-2 or 1amino acid residues are substituted, deleted or added, in anycombination.

The present invention further relates to polynucleotides that hybridizeto the herein above-described sequences. In this regard, the presentinvention especially relates to polynucleotides which hybridize understringent conditions to the herein above-described polynucleotides. Asherein used, the term "stringent conditions" means hybridization willoccur only if there is at least 80%, and preferably at least 90%, andmore preferably at least 95%, yet even more preferably 97-99% identitybetween the sequences.

Polynucleotides of the invention, which are identical or sufficientlyidentical to a nucleotide sequence contained in SEQ ID NO:1 or afragment thereof, may be used as hybridization probes for cDNA andgenomic DNA, to isolate full-length cDNAs and genomic clones encodingHNEAA81 and to isolate cDNA and genomic clones of other genes (includinggenes encoding homologs and orthologs from species other than human)that have a high sequence similarity to the HNEAA81 gene. Suchhybridization techniques are known to those of skill in the art.Typically these nucleotide sequences are 80% identical, preferably 90%identical, more preferably 95% identical to that of the referent. Theprobes generally will comprise at least 15 nucleotides. Preferably, suchprobes will have at least 30 nucleotides and may have at least 50nucleotides. Particularly preferred probes will range between 30 and 50nucleotides.

In one embodiment, to obtain a polynucleotide encoding the HNEAA81polypeptide, including homologs and orthologs from species other thanhuman, the method comprises screening an appropriate library understringent hybridization conditions with a labeled probe having the SEQID NO:1 or a fragment thereof; and isolating full-length cDNA andgenomic clones containing said polynucleotide sequence. Thus in anotheraspect, HNEAA81 polynucleotides of the present invention further includea nucleotide sequence comprising a nucleotide sequence that hybridizeunder stringent condition to a nucleotide sequence having SEQ ID NO:1 ora fragment thereof. Also included with HNEAA81 polypeptides arepolypeptides comprising amino acid sequences encoded by nucleotidesequences obtained by the above hybridization condition. Suchhybridization techniques are well known to those of skill in the art.Stringent hybridization conditions are as defmed above or,alternatively, conditions under overnight incubation at 42° C. in asolution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt's solution, 10%dextran sulfate, and 20 microgram/ml denatured, sheared salmon spermDNA, followed by washing the filters in 0.1×SSC at about 65° C.

The polynucleotides and polypeptides of the present invention may beemployed as research reagents and materials for discovery of treatmentsand diagnostics to animal and human disease.

Vectors, Host Cells, Expression

The present invention also relates to vectors which comprise apolynucleotide or polynucleotides of the present invention, and hostcells which are genetically engineered with vectors of the invention andto the production of polypeptides of the invention by recombinanttechniques. Cell-free translation systems can also be employed toproduce such proteins using RNAs derived from the DNA constructs of thepresent invention.

For recombinant production, host cells can be genetically engineered toincorporate expression systems or portions thereof for polynucleotidesof the present invention. Introduction of polynucleotides into hostcells can be effected by methods described in many standard laboratorymanuals, such as Davis, et al., BASIC METHODS IN MOLECULAR BIOLOGY(1986)and Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)such as calcium phosphate transfection, DEAE-dextran mediatedtransfection, transvection, microinjection, cationic lipid-mediatedtransfection, electroporation, transduction, scrape loading, ballisticintroduction or infection.

Representative examples of appropriate hosts include bacterial cells,such as streptococci, staphylococci, E. coli, Streptomyces and Bacillussubtilis cells; fungal cells, such as yeast cells and Aspergillus cells;insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animalcells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanomacells; and plant cells.

A great variety of expression systems can be used. Such systems include,among others, chromosomal, episomal and virus-derived systems, e.g.vectors derived from bacterial plasmids, from bacteriophage, fromtransposons, from yeast episomes, from insertion elements, from yeastchromosomal elements, from viruses such as baculoviruses, papovaviruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses,pseudorabies viruses and retroviruses, and vectors derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, such as cosmids and phagemids. Theexpression systems may contain control regions that regulate as well asengender expression. Generally, any system or vector suitable tomaintain, propagate or express polynucleotides to produce a polypeptidein a host may be used. The appropriate nucleotide sequence may beinserted into an expression system by any of a variety of well-known androutine techniques, such as, for example, those set forth in Sambrook,et al., MOLECULAR CLONING, A LABORATORY MANUAL (supra).

For secretion of the translated protein into the lumen of theendoplasmicreticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the desired polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals.

If the HNEAA81 polypeptide is to be expressed for use in screeningassays, generally, it is preferred that the polypeptide be produced atthe surface of the cell. In this event, the cells may be harvested priorto use in the screening assay. If the HNEAA81 polypeptide is secretedinto the medium, the medium can be recovered in order to recover andpurify the polypeptide; if produced intracellularly, the cells mustfirst be lysed before the polypeptide is recovered.

HNEAA81 polypeptides can be recovered and purified from recombinant cellcultures by well-known methods including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Most preferably, high performance liquid chromatographyis employed for purification. Well known techniques for refoldingproteins may be employed to regenerate active conformation when thepolypeptide is denatured during isolation and or purification.

Diagnostic Assays

This invention also relates to the use of HNEAA81 polynucleotides foruse as diagnostic reagents. Detection of a mutated form of the HNEAA81gene associated with a dysfunction will provide a diagnostic tool thatcan add to or define a diagnosis of a disease or susceptibility to adisease which results from under-expression, over-expression or alteredexpression of HNEAA81. Individuals carrying mutations in the HNEAA81gene may be detected at the DNA level by a variety of techniques.

Nucleic acids for diagnosis may be obtained from a subjects cells, suchas from blood, urine, saliva, tissue biopsy or autopsy material. Thegenomic DNA may be used directly for detection or may be amplifiedenzymatically by using PCR or other amplification techniques prior toanalysis. RNA or cDNA may also be used in similar fashion. Deletions andinsertions can be detected by a change in size of the amplified productin comparison to the normal genotype. Point mutations can be identifiedby hybridizing amplified DNA to labeled HNEAA81 nucleotide sequences.Perfectly matched sequences can be distinguished from mismatchedduplexes by RNase digestion or by differences in melting temperatures.DNA sequence differences may also be detected by alterations inelectrophoretic mobility of DNA fragments in gels, with or withoutdenaturing agents, or by direct DNA sequencing. See, e.g. Myers, et al.,Science (1985) 230:1242. Sequence changes at specific locations may alsobe revealed by nuclease protection assays, such as RNase and Slprotection or the chemical cleavage method. See Cotton, et al., ProcNatl Acad Sci USA (1985) 85: 4397-4401. In another embodiment, an arrayof oligonucleotide probes comprising the HNEAA81 nucleotide sequence orfragments thereof can be constructed to conduct efficient screening ofe.g., genetic mutations. Array technology methods are well known andhave general applicability and can be used to address a variety ofquestions in molecular genetics including gene expression, geneticlinkage, and genetic variability. (See, e.g., M.Chee, et al., Science,Vol 274, pp 610-613 (1996)).

The diagnostic assays offer a process for diagnosing or determining asusceptibility to infections such as bacterial, fungal, protozoan andviral infections, particularly infections caused by HIV-1 or HIV-2;pain; cancers; anorexia; bulimia; asthma; Parkinson's disease; acuteheart failure; hypotension; hypertension; urinary retention;osteoporosis; angina pectoris; myocardial infarction; ulcers; asthma;allergies; benign prostatic hypertrophy; and psychotic and neurologicaldisorders, including anxiety, schizophrenia, manic depression, delirium,dementia, severe mental retardation and dyskinesias, such asHuntington's disease or Gilles dela Tourett's syndrome, throughdetection of mutation in the HNEAA81 gene by the methods described.

In addition, infections such as bacterial, fungal, protozoan and viralinfections, particularly infections caused by HIV-1 or HIV-2; pain;cancers; anorexia; bulimia; asthma; Parkinson's disease; acute heartfailure; hypotension; hypertension; urinary retention; osteoporosis;angina pectoris; myocardial infarction; ulcers; asthma; allergies;benign prostatic hypertrophy; and psychotic and neurological disorders,including anxiety, schizophrenia, manic depression, delirium, dementia,severe mental retardation and dyskinesias, such as Huntington's diseaseor Gilles dela Tourett's syndrome, can be diagnosed by methodscomprising determining from a sample derived from a subject anabnormally decreased or increased level of the HNEAA81 polypeptide orHNEAA81 mRNA. Decreased or increased expression can be measured at theRNA level using any of the methods well known in the art for thequantitation of polynucleotides, such as, for example, PCR, RT-PCR,RNase protection, Northern blotting and other hybridization methods.Assay techniques that can be used to determine levels of a protein, suchas an HNEAA81, in a sample derived from a host are well-known to thoseof skill in the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis and ELISA assays.

Thus in another aspect, the present invention relates to a diagnostickit for a disease or susceptibility to a disease, particularlyinfections such as bacterial, fungal, protozoan and viral infections,particularly infections caused by HIV-1 or HIV-2; pain; cancers;anorexia; bulimia; asthma; Parkinson's disease; acute heart failure;hypotension; hypertension; urinary retention; osteoporosis; anginapectoris; myocardial infarction; ulcers; asthma; allergies;benignprostatic hypertrophy; and psychotic and neurological disorders,including anxiety, schizophrenia, manic depression, delirium, dementia,severe mental retardation and dyskinesias, such as Huntington's diseaseor Gilles dela Tourett's syndrome, which comprises:

(a) an HNEAA81 polynucleotide, preferably the nucleotide sequence of SEQID NO:1, or a fragment thereof;

(b) a nucleotide sequence complementary to that of (a);

(c) an HNEAA81 polypeptide, preferably the polypeptide of SEQ ID NO:2,or a fragment thereof; or

(d) an antibody to an HNEAA81 polypeptide, preferably to the polypeptideof SEQ ID NO: 2.

It will be appreciated that in any such kit, (a), (b), (c) or (d) maycomprise a substantial component.

Chromosome Assays

The nucleotide sequences of the present invention are also valuable forchromosome identification. The sequence is specifically targeted to andcan hybridize with a particular location on an individual humanchromosome. The mapping of relevant sequences to chromosomes accordingto the present invention is an important first step in correlating thosesequences with gene associated disease. Once a sequence has been mappedto a precise chromosomal location, the physical position of the sequenceon the chromosome can be correlated with genetic map data. Such data arefound, for example, in V. McKusick, Mendelian Inheritance in Man(available on line through Johns Hopkins University Welch MedicalLibrary). The relationship between genes and diseases that have beenmapped to the same chromosomal region are then identified throughlinkage analysis (coinheritance of physically adjacent genes).

The differences in the CDNA or genomic sequence between affected andunaffected individuals can also be determined. If a mutation is observedin some or all of the affected individuals but not in any normalindividuals, then the mutation is likely to be the causative agent ofthe disease. The gene of the present invention maps to human chromosome3q25.2.

Antibodies

The polypeptides of the invention or their fragments or analogs thereof,or cells expressing them can also be used as immunogens to produceantibodies immunospecific for the HNEAA81 polypeptides. The term"immunospecific" means that the antibodies have substantially greateraffinity for the polypeptides of the invention than their affinity forother related polypeptides in the prior art.

Antibodies generated against the HNEAA81 polypeptides can be obtained byadministering the polypeptides or epitope-bearing fragments, analogs orcells to an animal, preferably a nonhuman, using routine protocols. Forpreparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler, et al., Nature (1975)256:495-497), the trioma technique, the human B-cell hybridoma technique(Kozbor, et al., Immunology Today (1983) 4:72) and the EBV-hybridomatechnique (Cole, et al., MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp.77-96, Alan R. Liss, Inc., 1985).

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can also be adapted to produce single chain antibodies topolypeptides of this invention. Also, transgenic mice, or otherorganisms including other mammals, may be used to express humanizedantibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptide or to purify the polypeptides byaffinity chromatography.

Antibodies against HNEAA81 polypeptides may also be employed to treatinfections such as bacterial, fangal, protozoan and viral infections,particularly infections caused by HIV-1 or HIV-2; pain; cancers;anorexia; bulimia; asthma; Parkinson's disease; acute heartfailure;hypotension; hypertension; urinary retention; osteoporosis;angina pectoris; myocardial infarction; ulcers; asthma; allergies;benign prostatic hypertrophy; and psychotic and neurological disorders,including anxiety, schizophrenia, manic depression, delirium, dementia,severe mental retardation and dyskinesias, such as Huntington's diseaseor Gilles dela Tourett's syndrome, among others.

Vaccines

Another aspect of the invention relates to a method for inducing animmunological response in a mammal which comprises inoculating themammal with the HNEAA81 polypeptide, or a fragment thereof, adequate toproduce antibody and/or T cell immune response to protect said animalfrom infections such as bacterial, fungal, protozoan and viralinfections, particularly infections caused by HIV-1 or HIV-2; pain;cancers; anorexia; bulimia; asthma; Parkinson's disease; acute heartfailure; hypotension; hypertension; urinary retention; osteoporosis;angina pectoris; myocardial infarction; ulcers; asthma; allergies;benignprostatic hypertrophy; and psychotic and neurological disorders,including anxiety, schizophrenia, manic depression, delirium, dementia,severe mental retardation and dyskinesias, such as Huntington's diseaseor Gilles dela Tourett's syndrome, among others. Yet another aspect ofthe invention relates to a method of inducing immunological response ina mammal which comprises delivering the HNEAA8 1 polypeptide via avector directing expression of the HNEAA81 polynucleotide in vivo inorder to induce such an immunological response to produce antibody toprotect said animal from diseases.

A further aspect of the invention relates to an immunological/vaccineformulation (composition) which, when introduced into a mammalian host,induces an immunological response in that mammal to an HNEAA81polypeptide wherein the composition comprises an HNEAA81 polypeptide orHNEAA81 gene. The vaccine formulation may further comprise a suitablecarrier. Since HNEAA81 polypeptides may be broken down in the stomach,it is preferably administered parenterally (including subcutaneous,intramuscular, intravenous, intradermal, etc., injection). Formulationssuitable for parenteral administration include aqueous and non-aqueoussterile injection solutions which may contain anti-oxidants, buffers,bacteriostats and solutes which render the formulation isotonic with theblood of the recipient; and aqueous and non-aqueous sterile suspensionswhich may include suspending agents or thickening agents. Theformulations may be presented in unit-dose or multi-dose containers, forexample, sealed ampoules and vials and may be stored in a freeze-driedcondition requiring only the addition of the sterile liquid carrierimmediately prior to use. The vaccine formulation may also includeadjuvant systems for enhancing the immunogenicity of the formulation,such as oil-in water systems and other systems known in the art. Thedosage will depend on the specific activity of the vaccine and can bereadily determined by routine experimentation.

Screening Assays

The HNEMAA81 polypeptide of the present invention may be employed in aprocess for screening for compounds which bind to and activate theHNEAA81 polypeptides of the present invention (called agonists), orinhibit the interaction of the HNEAA81 polypeptides with receptorligands (called antagonists).

Thus, polypeptides of the invention may also be used to assess thebinding of small molecule substrates and ligands in, for example, cells,cell-free preparations, chemical libraries, and natural productmixtures. These substrates and ligands may be natural substrates andligands or may be structural or functional mimetics. See Coligan, etal., Current Protocols in Immunology 1(2):Chapter 5 (1991).

HNEAA81 proteins are responsible for many biological functions,including many pathologies. Accordingly, it is desirous to findcompounds and drugs which stimulate HNEAA81 on the one hand and whichcan inhibit the function of HNEAA81 on the other hand. In general,agonists are employed for therapeutic and prophylactic purposes for suchconditions as: infections such as bacterial, fungal, protozoan and viralinfections, particularly infections caused by HIV-1 or HIV-2; pain;cancers; anorexia; bulimia; asthma; Parkinson's disease; acute heartfailure; hypotension; hypertension; urinary retention; osteoporosis;angina pectoris; myocardial infarction; ulcers; asthma; allergies;benign prostatic hypertrophy; and psychotic and neurological disorders,including anxiety, schizophrenia, manic depression, delirium, dementia,severe mental retardation and dyskinesias, such as Huntington's diseaseor Gilles dela Tourett's syndrome, among others.

In general, such screening procedures involve providing appropriatecells which express the receptor polypeptide of the present invention onthe surface thereof. Such cells include cells from mammals, yeast,Drosophila or E. coli. In particular, a polynucleotide encoding thereceptor of the present invention is employed to transfect cells tothereby express the HNEAA81 polypeptide. The expressed receptor is thencontacted with a test compound to observe binding, stimulation orinhibition of a functional response.

One such screening procedure involves the use of melanophores which aretransfected to express the HNEAA81 polypeptide of the present invention.Such a screening technique is described in PCT WO 92/01810, publishedFeb. 6, 1992. Such an assay may be employed to screen for a compoundwhich inhibits activation of the receptor polypeptide of the presentinvention by contacting the melanophore cells which encode the receptorwith both the receptor ligand, such AP6A, AP5A or d-UDP, and a compoundto be screened. Inhibition of the signal generated by the ligandindicates that a compound is a potential antagonist for the receptor,i.e., inhibits activation of the receptor.

The technique may also be employed for screening of compounds whichactivate the receptor by contacting such cells with compounds to bescreened and determining whether such compound generates a signal, i.e.,activates the receptor.

Other screening techniques include the use of cells which express theHNEAA81 polypeptide (for example, transfected CHO cells) in a systemwhich measures extracellular pH changes caused by receptor activation.In this technique, compounds may be contacted with cells expressing thereceptor polypeptide of the present invention. A second messengerresponse, e.g., signal transduction or pH changes, is then measured todetermine whether the potential compound activates or inhibits thereceptor.

Another screening technique involves expressing the HNEAA81 polypeptidein which the receptor is linked to phospholipase C or D. Representativeexamples of such cells include, but are not limited to, endothelialcells, smooth muscle cells, and embryonic kidney cells. The screeningmay be accomplished as hereinabove described by detecting activation ofthe receptor or inhibition of activation of the receptor from thephospholipase second signal.

Another method involves screening for compounds which are antagonists,and thus inhibit activation of the receptor polypeptide of the presentinvention by determining inhibition of binding of labeled ligand, suchas AP6A, AP5A, or d-UDP, to cells which have the receptor on the surfacethereof, or cell membranes containing the receptor. Such a methodinvolves transfecting a eukaryotic cell with DNA encoding the HNEAA81polypeptide such that the cell expresses the receptor on its surface.The cell is then contacted with a potential antagonist in the presenceof a labeled form of a ligand, such as AP6A, AP5A, or d-UDP. The ligandcan be labeled, e.g., by radioactivity. The amount of labeled ligandbound to the receptors is measured, e.g., by measuring radioactivityassociated with transfected cells or membrane from these cells. If thecompound binds to the receptor, the binding of labeled ligand to thereceptor is inhibited as determined by a reduction of labeled ligandwhich binds to the receptors. This method is called binding assay.Naturally, this same technique can be used to look for an agonist.

Another screening procedure involves the use of mammalian cells (CHO,HEK 293, Xenopus Oocytes, RBL-2H3, etc.) which are transfected toexpress the receptor of interest. The cells are loaded with an indicatordye that produces a fluorescent signal when bound to calcium, and thecells are contacted with a test substance and a receptor agonist, suchas AP6A, AP5A, or d-UDP. Any change in fluorescent signal is measuredover a defined period of time using, for example, a fluorescencespectrophotometer or a fluorescence imaging plate reader. A change inthe fluorescence signal pattern generated by the ligand indicates that acompound is a potential antagonist or agonist for the receptor.

Another screening procedure involves use of mammalian cells (CHO,HEK293, Xenopus Oocytes, RBL-2H3, etc.) which are transfected to expressthe receptor of interest, and which are also transfected with a reportergene construct that is coupled to activation of the receptor (forexample, luciferase or beta-galactosidase behind an appropriatepromoter). The cells are contacted with a test substance and thereceptor agonist (ligand), such as AP6A, AP5A, or d-UDP, and the signalproduced by the reporter gene is measured after a defined period oftime. The signal can be measured using a luminometer, spectrophotometer,fluorimeter, or other such instrument appropriate for the specificreporter construct used. Inhibition of the signal generated by theligand indicates that a compound is a potential antagonist for thereceptor.

Another screening technique for antagonists or agonists involvesintroducing RNA encoding the HNEAA81 polypeptide into Xenopus oocytes(or CHO, HEK 293, RBL-2H3, etc.) to transiently or stably express thereceptor. The receptor oocytes are then contacted with the receptorligand, such as AP6A, AP5A, or D-UDP, and a compound to be screened.Inhibition or activation of the receptor is then determined by detectionof a signal, such as, cAMP, calcium, proton, or other ions.

Another method involves screening for HNEAA81 polypeptide inhibitors bydetermining inhibition or stimulation of HNEAA81 polypeptide-mediatedcAMP and/or adenylate cyclase accumulation or dimunition. Such a methodinvolves transiently or stably transfecting a eukaryotic cell withHNEAA81 polypeptide receptor to express the receptor on the cellsurface. The cell is then exposed to potential antagonists in thepresence of HNEAA81 polypeptide ligand, such as AP6A, AP5A, or d-UDP.The changes in levels of cAMP is then measured over a defined period oftime, for example, by radio-immuno or protein binding assays (forexample using Flashplates or a scintillation proximity assay). Changesin cAMP levels can also be determined by directly measuring the activityof the enzyme, adenylyl cyclase, in broken cell preparations. If thepotential antagonist binds the receptor, and thus inhibits HNEAA81polypeptide-ligand binding, the levels of HNEAA81 polypeptide-mediatedcAMP, or adenylate cyclase activity, will be reduced or increased.

Another screening method for agonists and antagonists relies on theendogenous pheromone response pathway in the yeast, Saccharomycescerevisiae. Heterothallic strains of yeast can exist in two mitoticallystable haploid mating types, MATa and MATa. Each cell type secretes asmall peptide hormone that binds to a G-protein coupled receptor onopposite mating-type cells which triggers a MAP kinase cascade leadingto G1 arrest as a prelude to cell fusion. Genetic alteration of certaingenes in the pheromone response pathway can alter the normal response topheromone, and heterologous expression and coupling of human G-proteincoupled receptors and humanized G-protein subunits in yeast cells devoidof endogenous pheromone receptors can be linked to downstream signalingpathways and reporter genes (e.g., U.S. Pat. Nos. 5,063,154; 5,482,835;5,691,188). Such genetic alterations include, but are not limited to,(i) deletion of the STE2 or STE3 gene encoding the endogenous G-proteincoupled pheromone receptors; (ii) deletion of the FAR1 gene encoding aprotein that normally associates with cyclin-dependent kinases leadingto cell cycle arrest; and (iii) construction of reporter genes fused tothe FUS1 gene promoter (where FUS1 encodes a membrane-anchoredglycoprotein required for cell fusion). Downstream reporter genes canpermit either a positive growth selection (e.g., histidine prototrophyusing the FUS1 -HIS3 reporter), or a colorimetric, fluorimetric orspectrophotometric readout, depending on the specific reporter constructused (e.g., b-galactosidase induction using a FUS1-LacZ reporter).

The yeast cells can be further engineered to express and secrete smallpeptides from random peptide libraries, some of which can permitautocrine activation of heterologously expressed human (or mammalian)G-protein coupled receptors (Broach, et al., Nature 384: 14-16, 1996;Manfredi, et al., Mol. Cell. Biol. 16: 4700-4709, 1996). This provides arapid direct growth selection (e.g., using the FUS1 -HIS3 reporter) forsurrogate peptide agonists that activate characterized or orphanreceptors. Alternatively, yeast cells that functionally express human(or mammalian) G-protein coupled receptors linked to a reporter genereadout (e.g., FUS1-LacZ) can be used as a platform for high-throughputscreening of known ligands, fractions of biological extracts andlibraries of chemical compounds for either natural or surrogate ligands.Functional agonists of sufficient potency (whether natural or surrogate)can be used as screening tools in yeast cell-based assays foridentifying G-protein coupled receptor antagonists. For example,agonists will promote growth of a cell with FUS-HIS3 reporter or givepositive readout for a cell with FUS1-LacZ. However, a candidatecompound which inhibits growth or negates the positive readout inducedby an agonist is an antagonist. For this purpose, the yeast systemoffers advantages over mammalian expression systems due to its ease ofutility and null receptor background (lack of endogenous G-proteincoupled receptors) which often interferes with the ability to identifyagonists or antagonists.

The present invention also provides a method for identifying new ligandsnot known to be capable of binding to an HNEAA81 polypeptides. Thescreening assays described above for identifying agonists may be used toidentify new ligands.

The present invention also contemplates agonists and antagonistsobtainable from the above described screening methods.

Examples of potential HNEAA81 polypeptide receptor antagonists includepeptidomimetics, synthetic organic molecules, natural products,antibodies, etc., which bind to the receptor, but do not elicit a secondmessenger response, such that the activity of the receptor is prevented.

Potential antagonists also include proteins which are closely related tothe ligand of the HNEAA81 polypeptide receptor, i.e., a fragment of theligand, which have lost biological function, and when they bind to theHNEAA81 polypeptide receptor, elicit no response.

Thus in another aspect, the present invention relates to a screening kitfor identifying agonists, antagonists, and ligands for HNEAA81polypeptides, which comprises:

(a) a HNEAA81 polypeptide, preferably that of SEQ ID NO:2; and furtherpreferably comprises labeled or unlabeled AP6A, AP5A, or d-UDP;

(b) a recombinant cell expressing a HNEAA81 polypeptide, preferably thatof SEQ ID NO:2; and further preferably comprises labeled or unlabeledAP6A, AP5A, or d-UDP; or

(c) a cell membrane expressing HNEAA81 polypeptide; preferably that ofSEQ ID NO: 2; and further preferably comprises labeled or unlabeledAP6A, AP5A, or d-UDP.

It will be appreciated that in any such kit, (a), (b), or (c) maycomprise a substantial component.

As noted above, a potential antagonist is a small molecule which bindsto the HNEAA81 polypeptide receptor, making it inaccessible to ligandssuch that normal biological activity is prevented. Examples of smallmolecules include, but are not limited to, small peptides orpeptide-like molecules.

Potential antagonists also include soluble forms of HNEAA81 polypeptidereceptor, e.g., fragments of the receptor, which bind to the ligand andprevent the ligand from interacting with membrane bound HNEAA81polypeptide receptors.

The HNEAA81 polypeptide of the present invention may be employed in ascreening process for compounds which bind the receptor and whichactivate (agonists) or inhibit activation of (antagonists) the receptorpolypeptide of the present invention. Thus, polypeptides of theinvention may also be used to assess the binding of small moleculesubstrates and ligands in, for example, cells, cell-free preparations,chemical libraries, and natural product mixtures. These substrates andligands may be natural substrates and ligands or may be structural orfunctional mimetics. See Coligan, et al., Current Protocols inImmunology 1(2):Chapter 5 (1991).

HNEAA81 polypeptides are responsible for many biological functions,including many pathologies. Accordingly, it is desirous to findcompounds and drugs which stimulate HNEAA81 on the one hand and whichcan inhibit the function of HNEAA81 on the other hand. In general,agonists are employed for therapeutic and prophylactic purposes for suchconditions as: infections such as bacterial, fungal, protozoan and viralinfections, particularly infections caused by HIV-1 or HIV-2; pain;cancers; anorexia; bulimia; asthma; Parkinson's disease; acute heartfailure; hypotension; hypertension; urinary retention; osteoporosis;angina pectoris; myocardial infarction; ulcers; asthma; allergies;benign prostatic hypertrophy; and psychotic and neurological disorders,including anxiety, schizophrenia, manic depression, delirium, dementia,severe mental retardation and dyskinesias, such as Huntington's diseaseor Gilles dela Tourett's syndrome, among others.

In general, such screening procedures involve producing appropriatecells which express the receptor polypeptide of the present invention onthe surface thereof. Such cells include cells from mammals, yeast,Drosophila or E. coli. Cells expressing the receptor (or cell membranecontaining the expressed receptor) are then contacted with a testcompound to observe binding, or stimulation or inhibition of afunctional response.

One screening technique includes the use of cells which express receptorof this invention (for example, transfected CHO cells) in a system whichmeasures extracellular pH or intracellular calcium changes caused byreceptor activation. In this technique, compounds may be contacted withcells expressing the receptor polypeptide of the present invention. Asecond messenger response, e.g., signal transduction, pH changes, orchanges in calcium level, is then measured to determine whether thepotential compound activates or inhibits the receptor.

Another method involves screening for receptor inhibitors by determininginhibition or stimulation of receptor-mediated cAMP and/or adenylatecyclase accumulation. Such a method involves transfecting a eukaryoticcell with the receptor of this invention to express the receptor on thecell surface. The cell is then exposed to potential antagonists in thepresence of the receptor of this invention. The amount ofcAMPaccumulation is then measured. If the potential antagonist binds thereceptor, and thus inhibits receptor binding, the levels ofreceptor-mediated cAMP, or adenylate cyclase, activity will be reducedor increased. Another method for detecting agonists or antagonists forthe receptor of the present invention is the yeast based technology asdescribed in U.S. Pat. No. 5,482,835.

Prophylactic and Therapeutic Methods

This invention provides methods of treating abnormal conditions such as,infections such as bacterial, fungal, protozoan and viral infections,particularly infections caused by HIV-1 or HIV-2; pain; cancers;anorexia; bulimia; asthma; Parkinson's disease; acute heartfailure;hypotension; hypertension; urinary retention; osteoporosis;angina pectoris; myocardial infarction; ulcers; asthma; allergies;benign prostatic hypertrophy; and psychotic and neurological disorders,including anxiety, schizophrenia, manic depression, delirium, dementia,severe mental retardation and dyskinesias, such as Huntington's diseaseor Gilles dela Tourett's syndrome, related to both an excess of, andinsufficient amounts of, HNEAA81 activity.

If the activity of HNEAA81 is in excess, several approaches areavailable. One approach comprises administering to a subject aninhibitor compound (antagonist) as hereinabove described along with apharmaceutically acceptable carrier in an amount effective to inhibitactivation by blocking binding of ligands to the HNEAA81, or byinhibiting a second signal, and thereby alleviating the abnormalcondition. In another approach, soluble forms of HNEAA81 polypeptidesstill capable of binding the ligand in competition with endogenousHNEAA81 may be administered. Typical embodiments of such competitorscomprise fragments of the HNEAA81 polypeptide.

In still another approach, expression of the gene encoding endogenousHNEAA81 can be inhibited using expression blocking techniques. Knownsuch techniques involve the use of antisense sequences, eitherinternally generated or separately administered. See, for example,O'Connor, J Neurochem (1991) 56:560 in Oligodeoxynucleotides asAntisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.(1988). Alternatively, oligonucleotides which form triple helices withthe gene can be supplied. See, e.g., Lee, et al., Nucleic Acids Res.(1979) 6:3073; Cooney, et al., Science (1988) 241:456; Dervan, et al.,Science (1991) 251:1360. These oligomers can be administered per se orthe relevant oligomers can be expressed in vivo.

For treating abnormal conditions related to an under-expression ofHNEAA81 and its activity, several approaches are also available. Oneapproach comprises administering to a subject a therapeuticallyeffective amount of a compound which activates HNEAA81, i.e., an agonistas described above, in combination with a pharmaceutically acceptablecarrier, to thereby alleviate the abnormal condition. Alternatively,gene therapy may be employed to effect the endogenous production ofHNEAA81 by the relevant cells in the subject. For example, apolynucleotide of the invention may be engineered for expression in areplication defective retroviral vector, as discussed above. Theretroviral expression construct may then be isolated and introduced intoa packaging cell transduced with a retroviral plasmid vector containingRNA encoding a polypeptide of the present invention such that thepackaging cell now produces infectious viral particles containing thegene of interest. These producer cells may be administered to a subjectfor engineering cells in vivo and expression of the polypeptide in vivo.For overview of gene therapy, see Chapter 20, Gene Therapy and otherMolecular Genetic-based Therapeutic Approaches, (and references citedtherein) in Human Molecular Genetics, T Strachan and A P Read, BIOSScientific Publishers Ltd. (1996). Another approach is to administer atherapeutic amount of HNEAA81 polypeptides in combination with asuitable pharmaceutical carrier.

Formulation and Administration

Peptides, such as the soluble form of HNEAA81 polypeptides, and agonistsand antagonist peptides or small molecules, may be formulated incombination with a suitable pharmaceutical carrier. Such formulationscomprise a therapeutically effective amount of the polypeptide orcompound, and a pharmaceutically acceptable carrier or excipient. Suchcarriers include but are not limited to, saline, buffered saline,dextrose, water, glycerol, ethanol, and combinations thereof.Formulation should suit the mode of administration, and is well withinthe skill of the art. The invention further relates to pharmaceuticalpacks and kits comprising one or more containers filled with one or moreof the ingredients of the aforementioned compositions of the invention.

Polypeptides and other compounds of the present invention may beemployed alone or in conjunction with other compounds, such astherapeutic compounds.

Preferred forms of systemic administration of the pharmaceuticalcompositions include injection, typically by intravenous injection.Other injection routes, such as subcutaneous, intramuscular, orintraperitoneal, can be used. Alternative means for systemicadministration include transmucosal and transdermal administration usingpenetrants such as bile salts or fusidic acids or other detergents. Inaddition, if properly formulated in enteric or encapsulatedformulations, oral administration may also be possible. Administrationof these compounds may also be topical and/or localized, in the form ofsalves, pastes, gels and the like.

The dosage range required depends on the choice of peptide, the route ofadministration, the nature of the formulation, the nature of thesubject's condition, and the judgment of the attending practitioner.Suitable dosages, however, are in the range of 0.1-100 μg/kg of subject.Wide variations in the needed dosage, however, are to be expected inview of the variety of compounds available and the differingefficiencies of various routes of administration. For example, oraladministration would be expected to require higher dosages thanadministration by intravenous injection. Variations in these dosagelevels can be adjusted using standard empirical routines foroptimization, as is well understood in the art.

Polypeptides used in treatment can also be generated endogenously in thesubject, in treatment modalities often referred to as "gene therapy" asdescribed above. Thus, for example, cells from a subject may beengineered with a polynucleotide, such as a DNA or RNA, to encode apolypeptide ex vivo, and for example, by the use of a retroviral plasmidvector. The cells are then introduced into the subject.

EXAMPLE 1 Mammalian Cell Expression

The receptors of the present invention are expressed in either humanembryonic kidney 293 (HEK293) cells or adherent dhfr CHO cells. Tomaximize receptor expression, typically all 5' and 3' untranslatedregions (UTRs) are removed from the receptor cDNA prior to insertioninto a pCDN or pCDNA3 vector. The cells are transfected with individualreceptor cDNAs by lipofectin and selected in the presence of 400 mg/mlG418. After 3 weeks of selection, individual clones are picked andexpanded for further analysis. HEK293 or CHO cells transfected with thevector alone serve as negative controls. To isolate cell lines stablyexpressing the individual receptors, about 24 clones are typicallyselected and analyzed by Northern blot analysis. Receptor mRNAs aregenerally detectable in about 50% of the G418-resistant clones analyzed.

EXAMPLE 2 Ligand Bank for Binding and Functional Assays

A bank of over 200 putative receptor ligands has been assembled forscreening. The bank comprises: transmitters, hormones and chemokinesknown to act via a human seven transmembrane (7TM) receptor; naturallyoccurring compounds which may be putative agonists for a human 7TMreceptor, non-mammalian, biologically active peptides for which amammalian counterpart has not yet been identified; and compounds notfound in nature, but which activate 7TM receptors with unknown naturalligands. This bank is used to initially screen the receptor for knownligands, using both functional (i.e., calcium, cAMP, microphysiometer,oocyte electrophysiology, etc., see below) as well as binding assays.

EXAMPLE 3 Ligand Binding Assays

Ligand binding assays provide a direct method for ascertaining receptorpharmacology and are adaptable to a high throughput format. The purifiedligand for a receptor is radiolabeled to high specific activity (50-2000Ci/mmol) for binding studies. A determination is then made that theprocess of radiolabeling does not diminish the activity of the ligandtowards its receptor. Assay conditions for buffers, ions, pH and othermodulators such as nucleotides are optimized to establish a workablesignal to noise ratio for both membrane and whole cell receptor sources.For these assays, specific receptor binding is defined as totalassociated radioactivity minus the radioactivity measured in thepresence of an excess of unlabeled competing ligand. Where possible,more than one competing ligand is used to define residual nonspecificbinding.

EXAMPLE 4 Functional Assay in Xenopus Oocytes

Capped RNA transcripts from linearized plasmid templates encoding thereceptor cDNAs of the invention are synthesized in vitro with RNApolymerases in accordance with standard procedures. In vitro transcriptsare suspended in water at a final concentration of 0.2 mg/ml. Ovarianlobes are removed from adult female toads, Stage V defolliculatedoocytes are obtained, and RNA transcripts (10 ng/oocyte) are injected ina 50 nl bolus using a microinjection apparatus. Two electrode voltageclamps are used to measure the currents from individual Xenopus oocytesin response to agonist exposure. Recordings are made in Ca2+free Barth'smedium at room temperature. The Xenopus system can be used to screenknown ligands and tissue/cell extracts for activating ligands.

EXAMPLE 5 Microphysiometric Assays

Activation of a wide variety of secondary messenger systems results inextrusion of small amounts of acid from a cell. The acid formed islargelyas a result of the increased metabolic activity required to fuelthe intracellular signaling process. The pH changes in the mediasurrounding the cell are very small but are detectable by the CYTOSENSORmicrophysiometer (Molecular Devices Ltd., Menlo Park, Calif.). TheCYTOSENSOR is thus capable of detecting the activation of a receptorwhich is coupled to an energy utilizing intracellular signaling pathwaysuch as the G-protein coupled receptor of the present invention.

EXAMPLE 6 Extract/Cell Supernatant Screening

A large number of mammalian receptors exist for which there remains, asyet, no cognate activating ligand (agonist). Thus, active ligands forthese receptors may not be included within the ligand banks asidentified to date. Accordingly, the 7TM receptor of the invention isalso functionally screened (using calcium, cAMP, microphysiometer,oocyte electrophysiology, etc., functional screens) against tissueextracts to identify natural ligands. Extracts that produce positivefunctional responses can be sequentially subfractionated until anactivating ligand is isolated and identified.

EXAMPLE 7 Calcium and cAMP Functional Assays

7TM receptors which are expressed in HEK 293 cells have been shown to becoupled functionally to activation of PLC and calcium mobilizationand/or cAMP stimulation or inhibition. Basal calcium levels in the HEK293 cells in receptor-transfected or vector control cells were observedto be in the normal, 100 nM to 200 nM, range. HEK 293 cells expressingrecombinant receptors are loaded with fura 2 and in a single day greaterthan 150 selected ligands or tissue/cell extracts are evaluated foragonist induced calcium mobilization. Similarly, HEK 293 cellsexpressing recombinant receptors are evaluated for the stimulation orinhibition of cAMP production using standard cAMP quantitation assays.Agonists presenting a calcium transient or cAMP fluctuation are testedin vector control cells to determine if the response is unique to thetransfected cells expressing receptor.

EXAMPLE 8 HNEAA81 Receptor Ligand Discovery

HEK-293 cells were transiently co-transfected with a mammalianexpression plasmid encoding HNEAA81 polypeptide, along with cDNAsencoding either the promiscuous G-protein Ga16 or the chimericG-proteins Gqi5 or Gqo5and assayed on FLIPR (Fluorometric Imaging PlateReader) for a calcium mobilisation response following addition of AP6Aor AP5A or d-UDP.

A dose-dependent (EC50s˜300 nM), calcium mobilization response wasdetected following addition of AP6A (response with AP5A or d-UDP not sostrong) to cells transfected with HNEAA81 and the G-proteins. Theagonist nucleotides did not stimulate a calcium mobilization response inHEK-293 cells transfected only with HNEAA81, nor was a response detectedto these ligands in HEK-293 cells transfected only with Ga 16 orGqi5/Gqo5. The cDNAs for both the receptor and the G-proteins had to beexpressed in the HEK-293 in order to detect a functional response tothese agonists.

Additional G-protein must be present HEK-293 cells in order to detectcalcium signalling mediated through HNEAA81. Thus, in the case of usingHEK-293, as described above, additional G-protein(s) is (are) requiredto run screens for agonists and antagonists. It is possible that HNEAA81expressed in another cell, for example RBL-2H3, may signal throughcalcium pathways without requiring additional G-protein, as has beennoted for the C5a receptor (Martino, et al., J Biol. Chem. 1994269:14446-14450), which in some cells also requires additionalG-proteins.

All publications including, but not limited to, patents and patentapplications, cited in this specification, are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

The above description fully discloses the invention, including preferredembodiments thereof. Modifications and improvements of the embodimentsspecifically disclosed herein are within the scope of the followingclaims. Without further elaboration, it is believed that one skilled inthe art can, using the preceding description, utilize the presentinvention to its fullest extent. Therefore, the examples provided hereinare to be construed as merely illustrative and are not a limitation ofthe scope of the present invention in any way. The embodiments of theinvention in which an exclusive property or privilege is claimed aredefined as follows.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 2                                             - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1124 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - TCTGGTTTTT AAAAAATAGC ATTTGAAAAT CATGAAGGGC TTTTTGTTTT CT - #TTTGTTTG         60                                                                          - TATATATGTT TATTGGTAAC AGGTGACACT GGAAGCAATG AACACCACAG TG - #ATGCAAGG        120                                                                          - CTTCAACAGA TCTGAGCGGT GCCCCAGAGA CACTCGGATA GTACAGCTGG TA - #TTCCCAGC        180                                                                          - CCTCTACACA GTGGTTTTCT TGACCGGCAT CCTGCTGAAT ACTTTGGCTC TG - #TGGGTGTT        240                                                                          - TGTTCACATC CCCAGCTCCT CCACCTTCAT CATCTACCTC AAAAACACTT TG - #GTGGCCGA        300                                                                          - CTTGATAATG ACACTCATGC TTCCTTTCAA AATCCTCTCT GACTCACACC TG - #GCACCCTG        360                                                                          - GCAGCTCAGA GCTTTTGTGT GTCGTTTTTC TTCGGTGATA TTTTATGAGA CC - #ATGTATGT        420                                                                          - GGGCATCGTG CTGTTAGGGC TCATAGCCTT TGACAGATTC CTCAAGATCA TC - #AGACCTTT        480                                                                          - GAGAAATATT TTTCTAAAAA AACCTGTTTT TGCAAAAACG GTCTCAATCT TC - #ATCTGGTT        540                                                                          - CTTTTTGTTC TTCATCTCCC TGCCAAATAC GATCTTGAGC AACAAGGAAG CA - #ACACCATC        600                                                                          - GTCTGTGAAA AAGTGTGCTT CCTTAAAGGG GCCTCTGGGG CTGAAATGGC AT - #CAAATGGT        660                                                                          - AAATAACATA TGCCAGTTTA TTTTCTGGAC TGTTTTTATC CTAATGCTTG TG - #TTTTATGT        720                                                                          - GGTTATTGCA AAAAAAGTAT ATGATTCTTA TAGAAAGTCC AAAAGTAAGG AC - #AGAAAAAA        780                                                                          - CAACAAAAAG CTGGAAGGCA AAGTATTTGT TGTCGTGGCT GTCTTCTTTG TG - #TGTTTTGC        840                                                                          - TCCATTTCAT TTTGCCAGAG TTCCATATAC TCACAGTCAA ACCAACAATA AG - #ACTGACTG        900                                                                          - TAGACTGCAA AATCAACTGT TTATTGCTAA AGAAACAACT CTCTTTTTGG CA - #GCAACTAA        960                                                                          - CATTTGTATG GATCCCTTAA TATACATATT CTTATGTAAA AAATTCACAG AA - #AAGCTACC       1020                                                                          - ATGTATGCAA GGGAGAAAGA CCACAGCATC AAGCCAAGAA AATCATAGCA GT - #CAGACAGA       1080                                                                          #                 112 - #4CTGTACAT AGGGTTAACT TCTA                            - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 333 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Met Asn Thr Thr Val Met Gln Gly Phe Asn Ar - #g Ser Glu Arg Cys Pro         #                15                                                           - Arg Asp Thr Arg Ile Val Gln Leu Val Phe Pr - #o Ala Leu Tyr Thr Val         #            30                                                               - Val Phe Leu Thr Gly Ile Leu Leu Asn Thr Le - #u Ala Leu Trp Val Phe         #        45                                                                   - Val His Ile Pro Ser Ser Ser Thr Phe Ile Il - #e Tyr Leu Lys Asn Thr         #    60                                                                       - Leu Val Ala Asp Leu Ile Met Thr Leu Met Le - #u Pro Phe Lys Ile Leu         #80                                                                           - Ser Asp Ser His Leu Ala Pro Trp Gln Leu Ar - #g Ala Phe Val Cys Arg         #                95                                                           - Phe Ser Ser Val Ile Phe Tyr Glu Thr Met Ty - #r Val Gly Ile Val Leu         #           110                                                               - Leu Gly Leu Ile Ala Phe Asp Arg Phe Leu Ly - #s Ile Ile Arg Pro Leu         #       125                                                                   - Arg Asn Ile Phe Leu Lys Lys Pro Val Phe Al - #a Lys Thr Val Ser Ile         #   140                                                                       - Phe Ile Trp Phe Phe Leu Phe Phe Ile Ser Le - #u Pro Asn Thr Ile Leu         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ser Asn Lys Glu Ala Thr Pro Ser Ser Val Ly - #s Lys Cys Ala Ser Leu         #               175                                                           - Lys Gly Pro Leu Gly Leu Lys Trp His Gln Me - #t Val Asn Asn Ile Cys         #           190                                                               - Gln Phe Ile Phe Trp Thr Val Phe Ile Leu Me - #t Leu Val Phe Tyr Val         #       205                                                                   - Val Ile Ala Lys Lys Val Tyr Asp Ser Tyr Ar - #g Lys Ser Lys Ser Lys         #   220                                                                       - Asp Arg Lys Asn Asn Lys Lys Leu Glu Gly Ly - #s Val Phe Val Val Val         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Ala Val Phe Phe Val Cys Phe Ala Pro Phe Hi - #s Phe Ala Arg Val Pro         #               255                                                           - Tyr Thr His Ser Gln Thr Asn Asn Lys Thr As - #p Cys Arg Leu Gln Asn         #           270                                                               - Gln Leu Phe Ile Ala Lys Glu Thr Thr Leu Ph - #e Leu Ala Ala Thr Asn         #       285                                                                   - Ile Cys Met Asp Pro Leu Ile Tyr Ile Phe Le - #u Cys Lys Lys Phe Thr         #   300                                                                       - Glu Lys Leu Pro Cys Met Gln Gly Arg Lys Th - #r Thr Ala Ser Ser Gln         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Glu Asn His Ser Ser Gln Thr Asp Asn Ile Th - #r Leu Gly                     #               330                                                           __________________________________________________________________________

What is claimed is:
 1. An isolated polypeptide consisting of the aminoacid sequence of SEQ ID NO:2.
 2. An isolated polypeptide comprising theamino acid sequence of SEQ ID NO:2.